Quantitative PCR

Quantitative PCR reaction setup is tedious and time consuming. Automation of reaction setup eliminates both human error and contamination associated with manual processing. Sigma-Aldrich offers a full range of automation-compatible, high-throughput Quantitative PCR (QPCR) products for use with either probe-based or SYBR® Green based applications to provide accurate real-time quantification of DNA or RNA templates. These products are conveniently packaged as a 2x ReadyMix™ reagent to include everything necessary for QPCR, leaving only the addition of primers, template, and in the case of probe-specific mixes, fluorescent detection chemistry. Each ReadyMix contains Sigma’s antibody mediated hot start mechanism, JumpStart™ Taq DNA Polymerase, for highly specific amplification.

Walk-away automation of reaction setup allows you to concentrate on other aspects of your work.

Simplified Handling

Products are packaged as a 2x master mix to include everything necessary for QPCR, leaving only the addition of primers, template, and in the case of probe-specific mixes, fluorescent detection chemistry.

Dual labeled probes and molecular beacons are available from Sigma Custom Products. Click here for more information.

Performance Characteristics

Amplification of Human Genomic DNA Samples

Figure 1. A PCR master mix containing JumpStart Taq ReadyMix reagent, GAPDH forward and reverse primers, and probe set was added to dilutions of human genomic DNA using the automated method for the Biomek FX Liquid Handling Workstation. GAPDH specific primers were designed to produce a 284 bp amplicon. The dual labeled probe was designed with a FAM reporter and a dark hole quencher. Initial template copy number was 9,000 and diluted 10-fold in subsequent wells. Amplification was carried out for the following concentrations of DNA in 12 replicates: 150 ng/µl, 15 ng/µl, 1.5 ng/µl, 150 pg/µl, and 15 pg/µl, as pictured here from left to right. Reactions were carried out on the ABI PRISM® 7700 Sequence Detection Systems.

Cross-Contamination Analysis

Figure 2. To test for cross contamination, a method was developed in which two different master mixes were dispensed across a 96-well plate to alternating wells. The first master mix contained the JumpStart Taq ReadyMix reagent, human genomic DNA (1.5 ng/µl), GAPDH primers, and probe set. The second mix included everything in the first mix except the genomic DNA. All samples were subjected to amplification on the ABI PRISM 7700 Sequence Detection Systems. The numbers shown in above table indicate the values of Cycle Threshold (Ct) in each well. The wells without genomic DNA (in yellow) showed no evidence of amplified PCR products, demonstrating that no cross contamination is present.

Figure 3. A PCR master mix containing SYBR Green JumpStart Taq ReadyMix reagent and GAPDH forward and reverse primers was added to dilutions of human genomic DNA template using the automated method for the Biomek FX Liquid Handling Workstation. GAPDH specific primers were designed to produce a 284 bp amplicon. Initial template copy number was 1,200 and diluted 5-fold in subsequent wells. Amplification was carried out for the following concentrations of DNA in 12 replicates: 2 ng/µl, 0.4 ng/µl, 80 pg/µl, 16 pg/µl, and 3.2 pg/µl, as pictured here from left to right. Reactions were carried out on the ABI PRISM 7700 Sequence Detection Systems.

Figure 4. A PCR master mix containing SYBR Green Taq ReadyMix reagent for Quantitative RT-PCR, GAPDH forward and reverse primers was added to dilutions of human total RNA template using the automated method for the Biomek FX Liquid Handling Workstation. GAPDH specific primers were designed to produce a 284 bp amplicon. Amplification was carried out for the following concentrations of total RNA in 12 replicates: 50 ng/µl, 5 ng/µl, 500 pg/µl, 50 pg/µl, and 5 pg/µl, as pictured here from left to right. Reactions were carried out on the ABI PRISM 7700 Sequence Detection Systems.